Methane-oxidizing bacteria (methanotrophs) are capable of growth on methane as their sole source of carbon and energy. They are ubiquitous in the natural environment and play a significant role in carbon cycling in specific habitats. Since they are net consumers of volatile carbon, they have a direct impact on the quality of water systems. In addition, they are also capable of cooxidizing a variety of non-growth substrates including straight-chain and cyclic hydrocarbons, as well as halogenated hydrocarbons. Since many of these compounds are toxic, methanotrophs may play an important role in detoxification in nature, and may have potential for the development of detoxification processes for polluted water or toxic wastes. In order to understand the role that methanotrophs play in maintaining a healthy environment it is important to study this group in detail. However, studies of methanotrophic metabolism have been hampered in the past by the lack of genetic techniques. We have used recombinant DNA techniques to develop mutagenesis and gene transfer systems that are useful in a variety of methylotrophic bacteria, including methanotrophs. We propose to use these techniques to carry out a comprehensive study of transcriptional regulation of C-1 specific functions in methanotrophs. Using a combination of hybridization probes and mutants, we will isolate C-1 specific genes from three methanotrophs. These genes will be characterized physically and then used as tools to study transcriptional regulation in chemostat-grown cells. This combination of approaches should result in a definitive understanding of how key C-1 processes are regulated in methanotrophs.